The goal of this exploratory application is to establish a novel, highly effective, and extremely useful technique for gene silencing. This technique, once fully established, will offer a powerful tool for basic science research with application in clinical investigation. Toward this goal, we propose a systematic study under two specific aims. (1) Detailed analysis of targeted pre-mRNA modification and gene silencing using a yeast system Our preliminary experiments carried out in S. cerevisiae have demonstrated that RNA-guided 2'-O- methylation targeted to actin pre-mRNA at its branch point adenosine can inhibit actin pre-mRNA splicing, leading to a complete loss of actin mRNA and hence cell death. Building on this exciting result, we propose a systematic investigation in yeast, whereby we will perfect the technique. Specifically, two fundamental issues regarding gene silencing, namely target specificity and technique generalizability, will be further scrutinized. We will use microarrays and gene rescue by introducing an intron-less actin gene into the guide RNA- containing cells to address the substrate specificity issue. To address whether the technique can be generalized, we will test several additional intron-containing yeast genes. Furthermore, to provide a complete spectrum of all potential target sites in a pre-mRNA substrate, we plan to design guide RNAs to modify the other invariant nucleotides important for splicing. We believe that our systematic analysis will yield a reliable novel gene silencing technique in S. cerevisiae. (2) Targeted pre-mRNA modification and gene silencing in mammalian cells Building on our investigations in S. cerevisiae, we will apply our gene silencing technique to mammalian cells, whose genes, with few exceptions, contain a number of introns, many of which are involved in alternative splicing. Because the branch point nucleotide is difficult to identify upon simple inspection of a gene sequence (due to the loosely defined consensus sequence), we will use in vitro splicing systems to map the branch point nucleotide experimentally for each pre-mRNA of interest. Having identified the target nucleotide, we will then assess the effectiveness of our gene silencing method focusing on two interesting genes: Upf3X, involved in mRNA nonsense-mediated decay (NMD), and ERalpha involved in breast cancer. Both genes are extensively studied in our neighboring labs at the University of Rochester Medical Center, and reagents and assays, including RNAi knockdown assays, are available, offering an opportunity for a direct comparison between our method and the RNAi knockdown method. We believe that our novel method will prove effective and useful in mammalian systems as well as in yeast. [unreadable] [unreadable] [unreadable]